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Lignin valorization offers significant potential to enhance the economic viability of lignocellulosic biorefineries. However, because of its heterogeneous and recalcitrant nature, conversion of lignin to value-added coproducts remains a considerable technical challenge. In this study, we employ base-catalyzed depolymerization (BCD) using a process-relevant solid lignin stream produced via deacetylation, mechanical refining, and enzymatic hydrolysis to enable biological lignin conversion. BCD was conducted with the solid lignin substrate over a range of temperatures at two NaOH concentrations, and the results demonstrate that the lignin can be partially extracted and saponified at temperatures as low as 60 °C. At 120 °C and 2% NaOH, the high extent of lignin solubility was accompanied by a considerable decrease in the lignin average molecular weight and the release of lignin-derived monomers including hydroxycinnamic acids. BCD liquors were tested for microbial growth using seven aromatic-catabolizing bacteria and two yeasts. Three organisms (Pseudomonas putida KT2440, Rhodotorula mucilaginosa, and Corynebacterium glutamicum) tolerate high BCD liquor concentrations (up to 90% v/v) and rapidly consume the main lignin-derived monomers, resulting in lignin conversion of up to 15%. Furthermore, as a proof of concept, muconic acid production from a representative lignin BCD liquor was demonstrated with an engineered P. putida KT2440 strain. These results highlight the potential for a mild lignin depolymerization process to enhance the microbial conversion of solid lignin-rich biorefinery streams.

In Nature, powerful oxidative enzymes secreted by white rot fungi and some bacteria catalyze lignin depolymerization and some microbes are able to catabolize the resulting aromatic compounds as carbon and energy sources. Taken together, these two processes offer a potential route for microbial valorization of lignin. However, many challenges remain in realizing this concept, including that oxidative enzymes responsible for lignin depolymerization also catalyze polymerization of low molecular weight (LMW) lignin. Here, multiple basidiomycete secretomes were screened for ligninolytic enzyme activities in the presence of a residual lignin solid stream from a corn stover biorefinery, dubbed DMR-EH (Deacetylation, Mechanical Refining, and Enzymatic Hydrolysis) lignin. Two selected fungal secretomes, with high levels of laccases and peroxidases, were utilized for DMR-EH lignin depolymerization assays. The secretome from Pleurotus eryngii, which exhibited the highest laccase activity, reduced the lignin average molecular weight (Mw) by 63% and 75% at pH 7 compared to the Mw of the control treated at the same conditions and the initial DMR-EH lignin, respectively, and was applied in further depolymerization assays as a function of time. As repolymerization was observed after 3 days of incubation, an aromatic-catabolic microbe (Pseudomonas putida KT2440) was incubated with the fungal secretome and DMR-EH lignin. These experiments demonstrated that the presence of the bacterium enhances lignin depolymerization, likely due to bacterial catabolism of LMW lignin, which may partially prevent repolymerization. In addition, proteomics was also applied to the P. eryngii secretome to identify the enzymes present in the fungal cocktail utilized for the depolymerization assays, which highlighted a significant number of glucose/ methanol/choline (GMC) oxidoreductases and laccases. Overall, this study demonstrates that ligninolytic enzymes can be used to partially depolymerize a solid, high lignin content biorefinery stream and that the presence of an aromatic-catabolic bacterium as a "microbial sink" improves the extent of enzymatic lignin depolymerization.

Abstract Background In converting biomass to bioethanol, pretreatment is a key step intended to render cellulose more amenable and accessible to cellulase enzymes and thus increase glucose yields. In this study, four cellulose samples with different degrees of polymerization and crystallinity indexes were subjected to aqueous sodium hydroxide and anhydrous liquid ammonia treatments. The effects of the treatments on cellulose crystalline structure were studied, in addition to the effects on the digestibility of the celluloses by a cellulase complex. Results From X-ray diffractograms and nuclear magnetic resonance spectra, it was revealed that treatment with liquid ammonia produced the cellulose IIII allomorph; however, crystallinity depended on treatment conditions. Treatment at a low temperature (25°C) resulted in a less crystalline product, whereas treatment at elevated temperatures (130°C or 140°C) gave a more crystalline product. Treatment of cellulose I with aqueous sodium hydroxide (16.5 percent by weight) resulted in formation of cellulose II, but also produced a much less crystalline cellulose. The relative digestibilities of the different cellulose allomorphs were tested by exposing the treated and untreated cellulose samples to a commercial enzyme mixture (Genencor-Danisco; GC 220). The digestibility results showed that the starting cellulose I samples were the least digestible (except for corn stover cellulose, which had a high amorphous content). Treatment with sodium hydroxide produced the most digestible cellulose, followed by treatment with liquid ammonia at a low temperature. Factor analysis indicated that initial rates of digestion (up to 24 hours) were most strongly correlated with amorphous content. Correlation of allomorph type with digestibility was weak, but was strongest with cellulose conversion at later times. The cellulose IIII samples produced at higher temperatures had comparable crystallinities to the initial cellulose I samples, but achieved higher levels of cellulose conversion, at longer digestion times. Conclusions Earlier studies have focused on determining which cellulose allomorph is the most digestible. In this study we have found that the chemical treatments to produce different allomorphs also changed the crystallinity of the cellulose, and this had a significant effect on the digestibility of the substrate. When determining the relative digestibilities of different cellulose allomorphs it is essential to also consider the relative crystallinities of the celluloses being tested.

Feedstock variability represents a challenge in lignocellulosic biorefineries, as it can influence both lignocellulose deconstruction and microbial conversion processes for biofuels and biochemicals production. The impact of feedstock variability on microbial performance remains underexplored, and predictive tools for microbial behaviour are needed to mitigate risks in biorefinery scale-up. Here, twelve batches of corn stover were deconstructed via deacetylation, mechanical refining, and enzymatic hydrolysis to generate lignin-rich and sugar streams. These batches and their derived streams were characterised to identify their chemical components, and the streams were used as substrates for producing muconate and butyrate by engineered Pseudomonas putida and wildtype Clostridium tyrobutyricum, respectively. Bacterial performance (growth, product titers, yields, and productivities) differed among the batches, but no strong correlations were identified between feedstock composition and performance. To provide metabolic insights into the origin of these differences, we evaluated the effect of twenty-three isolated chemical components on these microbes, including three components in relevant bioprocess settings in bioreactors, and we found that growth-inhibitory concentrations were outside the ranges observed in the streams. Overall, this study generates a foundational dataset on P. putida and C. tyrobutyricum performance to enable future predictive models and underscores their resilience in effectively converting fluctuating lignocellulose-derived streams into bioproducts., (© 2024 The Author(s). Microbial Biotechnology published by John Wiley & Sons Ltd.)

Efforts to produce aromatic monomers through catalytic lignin depolymerization have historically focused on aryl-ether bond cleavage. A large fraction of aromatic monomers in lignin, however, are linked by various carbon-carbon (C-C) bonds that are more challenging to cleave and limit the yields of aromatic monomers from lignin depolymerization. Here, we report a catalytic autoxidation method to cleave C-C bonds in lignin-derived dimers and oligomers from pine and poplar. The method uses manganese and zirconium salts as catalysts in acetic acid and produces aromatic carboxylic acids as primary products. The mixtures of the oxygenated monomers are efficiently converted to cis,cis-muconic acid in an engineered strain of Pseudomonas putida KT2440 that conducts aromatic O-demethylation reactions at the 4-position. This work demonstrates that autoxidation of lignin with Mn and Zr offers a catalytic strategy to increase the yield of valuable aromatic monomers from lignin., (© 2024. The Author(s).)

Selective lignin depolymerization is a key step in lignin valorization to value-added products, and there are multiple catalytic methods to cleave labile aryl-ether bonds in lignin. However, the overall aromatic monomer yield is inherently limited by refractory carbon-carbon linkages, which are abundant in lignin and remain intact during most selective lignin deconstruction processes. In this work, we demonstrate that a Co/Mn/Br-based catalytic autoxidation method promotes carbon-carbon bond cleavage in acetylated lignin oligomers produced from reductive catalytic fractionation. The oxidation products include acetyl vanillic acid and acetyl vanillin, which are ideal substrates for bioconversion. Using an engineered strain of Pseudomonas putida , we demonstrate the conversion of these aromatic monomers to cis , cis -muconic acid. Overall, this study demonstrates that autoxidation enables higher yields of bioavailable aromatic monomers, exceeding the limits set by ether-bond cleavage alone., Competing Interests: The authors declare the following competing financial interest(s): N.X.G., C.T.P., K.P.S., S.S.S., and G.T.B. have filed a patent application on this work., (© 2023 The Authors. Published by American Chemical Society.)

Bioconversion of lignin-related aromatic compounds relies on robust catabolic pathways in microbes. Sphingobium sp. SYK-6 (SYK-6) is a well-characterized aromatic catabolic organism that has served as a model for microbial lignin conversion, and its utility as a biocatalyst could potentially be further improved by genome-wide metabolic analyses. To this end, we generate a randomly barcoded transposon insertion mutant (RB-TnSeq) library to study gene function in SYK-6. The library is enriched under dozens of enrichment conditions to quantify gene fitness. Several known aromatic catabolic pathways are confirmed, and RB-TnSeq affords additional detail on the genome-wide effects of each enrichment condition. Selected genes are further examined in SYK-6 or Pseudomonas putida KT2440, leading to the identification of new gene functions. The findings from this study further elucidate the metabolism of SYK-6, while also providing targets for future metabolic engineering in this organism or other hosts for the biological valorization of lignin., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Sphingobium sp. strain SYK-6 is an efficient aromatic catabolic bacterium that can consume all four stereoisomers of 1,2-diguaiacylpropane-1,3-diol (DGPD), which is a ring-opened β-1-type dimer. Recently, LdpA-mediated catabolism of erythro -DGPD was reported in SYK-6, but the catabolic pathway for threo -DGPD was as yet unknown. Here, we elucidated the catabolism of threo -DGPD, which proceeds through conversion to erythro -DGPD. When threo -DGPD was incubated with SYK-6, the Cα hydroxy groups of threo -DGPD (DGPD I and II) were initially oxidized to produce the Cα carbonyl form (DGPD-keto I and II). This initial oxidation step is catalyzed by Cα-dehydrogenases, which belong to the short-chain dehydrogenase/reductase (SDR) family and are involved in the catabolism of β-O-4-type dimers. Analysis of seven candidate genes revealed that NAD + -dependent LigD and LigL are mainly involved in the conversion of DGPD I and II, respectively. Next, we found that DGPD-keto I and II were reduced to erythro -DGPD (DGPD III and IV) in the presence of NADPH. Genes involved in this reduction were sought from Cα-dehydrogenase and ldpA -neighboring SDR genes. The gene products of SLG_12690 ( ldpC ) and SLG_12640 ( ldpB ) catalyzed the NADPH-dependent conversion of DGPD-keto I to DGPD III and DGPD-keto II to DGPD IV, respectively. Mutational analysis further indicated that ldpC and ldpB are predominantly involved in the reduction of DGPD-keto. Together, these results demonstrate that SYK-6 harbors a comprehensive catabolic enzyme system to utilize all four β-1-type stereoisomers through successive oxidation and reduction reactions of the Cα hydroxy group of threo -DGPD with a net stereoinversion using multiple dehydrogenases. IMPORTANCE In many catalytic depolymerization processes of lignin polymers, aryl-ether bonds are selectively cleaved, leaving carbon-carbon bonds between aromatic units intact, including dimers and oligomers with β-1 linkages. Therefore, elucidating the catabolic system of β-1-type lignin-derived compounds will aid in the establishment of biological funneling of heterologous lignin-derived aromatic compounds to value-added products. Here, we found that threo -DGPD was converted by successive stereoselective oxidation and reduction at the Cα position by multiple alcohol dehydrogenases to erythro -DGPD, which is further catabolized. This system is very similar to that developed to obtain enantiopure alcohols from racemic alcohols by artificially combining two enantiocomplementary alcohol dehydrogenases. The results presented here demonstrate that SYK-6 has evolved to catabolize all four stereoisomers of DGPD by incorporating this stereoinversion system into its native β-1-type dimer catabolic system., Competing Interests: The authors declare no conflict of interest.

Lignin valorization is being intensely pursued via tandem catalytic depolymerization and biological funneling to produce single products. In many lignin depolymerization processes, aromatic dimers and oligomers linked by carbon-carbon bonds remain intact, necessitating the development of enzymes capable of cleaving these compounds to monomers. Recently, the catabolism of erythro -1,2-diguaiacylpropane-1,3-diol ( erythro -DGPD), a ring-opened lignin-derived β-1 dimer, was reported in Novosphingobium aromaticivorans . The first enzyme in this pathway, LdpA (formerly LsdE), is a member of the nuclear transport factor 2 (NTF-2)-like structural superfamily that converts erythro -DGPD to lignostilbene through a heretofore unknown mechanism. In this study, we performed biochemical, structural, and mechanistic characterization of the N. aromaticivorans LdpA and another homolog identified in Sphingobium sp. SYK-6, for which activity was confirmed in vivo. For both enzymes, we first demonstrated that formaldehyde is the C 1 reaction product, and we further demonstrated that both enantiomers of erythro -DGPD were transformed simultaneously, suggesting that LdpA, while diastereomerically specific, lacks enantioselectivity. We also show that LdpA is subject to a severe competitive product inhibition by lignostilbene. Three-dimensional structures of LdpA were determined using X-ray crystallography, including substrate-bound complexes, revealing several residues that were shown to be catalytically essential. We used density functional theory to validate a proposed mechanism that proceeds via dehydroxylation and formation of a quinone methide intermediate that serves as an electron sink for the ensuing deformylation. Overall, this study expands the range of chemistry catalyzed by the NTF-2-like protein family to a prevalent lignin dimer through a cofactorless deformylation reaction.

Lignin is the largest source of bio-based aromatic compounds in nature, and its valorization is essential to the sustainability of lignocellulosic biorefining. Characterizing lignin-derived compounds remains challenging due to the heterogeneity of this biopolymer. Tandem mass spectrometry is a promising tool for lignin structural analytics, as fragmentation patterns of model compounds can be extrapolated to identify characteristic moieties in complex samples. This work extended previous resonance excitation-type collision-induced dissociation (CID) methods that identified lignin oligomers containing β-O-4, β-5, and β-β bonds, to also identify characteristics of 5-5, β-1, and 4-O-5 dimers, enabled by quadrupole time-of-flight (QTOF) CID with energy-resolved mass spectrometry (ERMS). Overall, QTOF-ERMS offers in-depth structural information and could ultimately contribute to tools for high-throughput lignin dimer identification., (© 2022 Wiley-VCH GmbH.)

Background: Commercial spray-dried egg white (S-EW) is often used for oral immunotherapy (OIT). Peptifine® (PF), a commercial preparation of enzymatically-hydrolyzed egg white (EW) peptides, is considered safe OIT food., Objective: Here, we examined the therapeutic effect of PF or S-EW in a mouse model of EW allergy., Methods: Sensitized female BALB/c mice underwent 4-week OIT by supplementation of 19% casein diet with 1% PF (PF group) or S-EW (S-EW group), and non-supplementation of 20% casein diet (non-OIT group). Non-sensitized mice acted as the non-allergy group. Oral and intraperitoneal EW challenges were performed and allergic biomarkers were determined., Results: Changes in rectal temperature after oral challenge were comparable in the allergy groups. However, after intraperitoneal challenge, rectal temperature decrease in the PF and S-EW groups was lower than in the non-OIT group. After 4 weeks, plasma levels of ovalbumin- and ovomucoid-specific antibodies were assessed; IgE levels in the PF and S-EW groups were lower than those in the non-OIT group. Interleukin-4 secretion after EW challenge was significantly lower in splenocyte cultures derived from PF and S-EW groups than in the non-OIT group. Furthermore, the percentage of CD4+ Foxp3+ splenocytes in the PF group was significantly higher than that in the non-OIT and S-EW groups. Oral gavage of 40 mg PF did not induce an allergic response in sensitized mice with EW., Conclusions: Diet supplementation with 1% PF mildly ameliorated the severity of allergy in mouse with EW allergy, indicating that PF is a safe OIT food.

Fragment-based drug discovery (FBDD), which involves small compounds <300 Da, has been recognized as one of the most powerful tools for drug discovery. In FBDD, the affinity of hit compounds tends to be low, and the analysis of protein-compound interactions becomes difficult. In an effort to overcome such difficulty, we developed a 19 F-NMR screening method optimizing a 19 F chemical library focusing on highly soluble monomeric molecules. Our method was successfully applied to four proteins, including protein kinases and a membrane protein. For FKBP12, hit compounds were carefully validated by protein thermal shift analysis, 1 H- 15 N HSQC NMR spectroscopy, and isothermal titration calorimetry to determine dissociation constants and model complex structures. It should be noted that the 1 H and 19 F saturation transfer difference experiments were crucial to obtaining highly precise model structures. The combination of 19 F-NMR analysis and the optimized 19 F chemical library enables the modeling of the complex structure made up of a weak binder and its target protein., Competing Interests: There are no conflicts of interest to declare., (This journal is © The Royal Society of Chemistry.)

Egg white (EW) allergy model mice underwent 15-day oral immunotherapy (OIT) using diets with different corn-oil content and 1% EW. After OIT, mortality following an oral food challenge in OIT groups was approximately 90%. However, in previous studies, mortality was 0%. This phenomenon is to be associated with increased corn-oil intake during allergy sensitization and allergen exposure for 2 weeks., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Lignin biosynthesis typically results in a polymer with several inter-monomer bond linkages, and the heterogeneity of linkages presents a challenge for depolymerization processes. While several enzyme classes have been shown to cleave common dimer linkages in lignin, the pathway of bacterial β-1 spirodienone linkage cleavage has not been elucidated. Here, we identified a pathway for cleavage of 1,2-diguaiacylpropane-1,3-diol (DGPD), a β-1 linked biaryl representative of a ring-opened spirodienone linkage, in Novosphingobium aromaticivorans DSM12444. In vitro assays using cell lysates demonstrated that RS14230 (LsdE) converts DGPD to a lignostilbene intermediate, which the carotenoid oxygenase, LsdA, then converts to vanillin. A Pseudomonas putida KT2440 strain engineered with lsdEA expression catabolizes erythro-DGPD, but not threo-DGPD. We further engineered P. putida to convert DGPD to a product, cis,cis-muconic acid. Overall, this work demonstrates the potential to identify new enzymatic reactions in N. aromaticivorans and expands the biological funnel of P. putida for microbial lignin valorization., (Copyright © 2021 International Metabolic Engineering Society. All rights reserved.)

Lignin is a biopolymer found in plant cell walls that accounts for 30% of the organic carbon in the biosphere. White-rot fungi (WRF) are considered the most efficient organisms at degrading lignin in nature. While lignin depolymerization by WRF has been extensively studied, the possibility that WRF are able to utilize lignin as a carbon source is still a matter of controversy. Here, we employ 13 C-isotope labeling, systems biology approaches, and in vitro enzyme assays to demonstrate that two WRF, Trametes versicolor and Gelatoporia subvermispora , funnel carbon from lignin-derived aromatic compounds into central carbon metabolism via intracellular catabolic pathways. These results provide insights into global carbon cycling in soil ecosystems and furthermore establish a foundation for employing WRF in simultaneous lignin depolymerization and bioconversion to bioproducts-a key step toward enabling a sustainable bioeconomy., Competing Interests: The authors declare no competing interest.

Lignostilbene-α,β-dioxygenases (LSDs) are iron-dependent oxygenases involved in the catabolism of lignin-derived stilbenes. Sphingobium sp. SYK-6 contains eight LSD homologs with undetermined physiological roles. To investigate which homologs are involved in the catabolism of dehydrodiconiferyl alcohol (DCA), derived from β-5 linked lignin subunits, we heterologously produced the enzymes and screened their activities in lysates. The seven soluble enzymes all cleaved lignostilbene, but only LSD2, LSD3, and LSD4 exhibited high specific activity for 3-(4-hydroxy-3-(4-hydroxy-3-methoxystyryl)-5-methoxyphenyl) acrylate (DCA-S) relative to lignostilbene. LSD4 catalyzed the cleavage of DCA-S to 5-formylferulate and vanillin and cleaved lignostilbene and DCA-S (∼10 6  M -1 s -1 ) with tenfold greater specificity than pterostilbene and resveratrol. X-ray crystal structures of native LSD4 and the catalytically inactive cobalt-substituted Co-LSD4 at 1.45 Å resolution revealed the same fold, metal ion coordination, and edge-to-edge dimeric structure as observed in related enzymes. Key catalytic residues, Phe-59, Tyr-101, and Lys-134, were also conserved. Structures of Co-LSD4·vanillin, Co-LSD4·lignostilbene, and Co-LSD4·DCA-S complexes revealed that Ser-283 forms a hydrogen bond with the hydroxyl group of the ferulyl portion of DCA-S. This residue is conserved in LSD2 and LSD4 but is alanine in LSD3. Substitution of Ser-283 with Ala minimally affected the specificity of LSD4 for either lignostilbene or DCA-S. By contrast, substitution with phenylalanine, as occurs in LSD5 and LSD6, reduced the specificity of the enzyme for both substrates by an order of magnitude. This study expands our understanding of an LSD critical to DCA catabolism as well as the physiological roles of other LSDs and their determinants of substrate specificity., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Cytochrome P450 enzymes have tremendous potential as industrial biocatalysts, including in biological lignin valorization. Here, we describe P450s that catalyze the O -demethylation of lignin-derived guaiacols with different ring substitution patterns. Bacterial strains Rhodococcus rhodochrous EP4 and Rhodococcus jostii RHA1 both utilized alkylguaiacols as sole growth substrates. Transcriptomics of EP4 grown on 4-propylguaiacol (4PG) revealed the up-regulation of agcA , encoding a CYP255A1 family P450, and the aph genes, previously shown to encode a meta -cleavage pathway responsible for 4-alkylphenol catabolism. The function of the homologous pathway in RHA1 was confirmed: Deletion mutants of agcA and aphC , encoding the meta -cleavage alkylcatechol dioxygenase, grew on guaiacol but not 4PG. By contrast, deletion mutants of gcoA and pcaL , encoding a CYP255A2 family P450 and an ortho -cleavage pathway enzyme, respectively, grew on 4-propylguaiacol but not guaiacol. CYP255A1 from EP4 catalyzed the O -demethylation of 4-alkylguaiacols to 4-alkylcatechols with the following apparent specificities ( k cat / K M ): propyl > ethyl > methyl > guaiacol. This order largely reflected AgcA's binding affinities for the different guaiacols and was the inverse of GcoA EP4 's specificities. The biocatalytic potential of AgcA was demonstrated by the ability of EP4 to grow on lignin-derived products obtained from the reductive catalytic fractionation of corn stover, depleting alkylguaiacols and alkylphenols. By identifying related P450s with complementary specificities for lignin-relevant guaiacols, this study facilitates the design of these enzymes for biocatalytic applications. We further demonstrated that the metabolic fate of the guaiacol depends on its substitution pattern, a finding that has significant implications for engineering biocatalysts to valorize lignin., Competing Interests: The authors declare no competing interest.

Lignin solvolysis from the plant cell wall is the critical first step in lignin depolymerization processes involving whole biomass feedstocks. However, little is known about the coupled reaction kinetics and transport phenomena that govern the effective rates of lignin extraction. Here, we report a validated simulation framework that determines intrinsic, transport-independent kinetic parameters for the solvolysis of lignin, hemicellulose, and cellulose upon incorporation of feedstock characteristics for the methanol-based extraction of poplar as an example fractionation process. Lignin fragment diffusion is predicted to compete on the same time and length scales as reactions of lignin within cell walls and longitudinal pores of typical milled particle sizes, and mass transfer resistances are predicted to dominate the solvolysis of poplar particles that exceed approximately 2 mm in length. Beyond the approximately 2 mm threshold, effectiveness factors are predicted to be below 0.25, which implies that pore diffusion resistances may attenuate observable kinetic rate measurements by at least 75 % in such cases. Thus, researchers are recommended to conduct kinetic evaluations of lignin-first catalysts using biomass particles smaller than approximately 0.2 mm in length to avoid feedstock-specific mass transfer limitations in lignin conversion studies. Overall, this work highlights opportunities to improve lignin solvolysis by genetic engineering and provides actionable kinetic information to guide the design and scale-up of emerging biorefinery strategies., (© 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Lignin is an abundant and recalcitrant component of plant cell walls. While lignin degradation in nature is typically attributed to fungi, growing evidence suggests that bacteria also catabolize this complex biopolymer. However, the spatiotemporal mechanisms for lignin catabolism remain unclear. Improved understanding of this biological process would aid in our collective knowledge of both carbon cycling and microbial strategies to valorize lignin to value-added compounds. Here, we examine lignin modifications and the exoproteome of three aromatic-catabolic bacteria: Pseudomonas putida KT2440, Rhodoccocus jostii RHA1, and Amycolatopsis sp. ATCC 39116. P. putida cultivation in lignin-rich media is characterized by an abundant exoproteome that is dynamically and selectively packaged into outer membrane vesicles (OMVs). Interestingly, many enzymes known to exhibit activity toward lignin-derived aromatic compounds are enriched in OMVs from early to late stationary phase, corresponding to the shift from bioavailable carbon to oligomeric lignin as a carbon source. In vivo and in vitro experiments demonstrate that enzymes contained in the OMVs are active and catabolize aromatic compounds. Taken together, this work supports OMV-mediated catabolism of lignin-derived aromatic compounds as an extracellular strategy for nutrient acquisition by soil bacteria and suggests that OMVs could potentially be useful tools for synthetic biology and biotechnological applications., Competing Interests: The authors declare no competing interest.

The self-localizing ligand-induced protein translocation (SLIPT) system is an emerging platform that controls protein localization in living cells using synthetic self-localizing ligands (SLs). Here, we report a chemogenetic SLIPT system for inducing protein translocation from the cytoplasm to the surface of the endoplasmic reticulum (ER) and Golgi membranes, referred to as endomembranes. By screening a series of lipid-trimethoprim (TMP) conjugates, we found oleic acid-tethered TMP (oleTMP) to be the optimal SL that efficiently relocated and anchored Escherichia coli dihydrofolate reductase (eDHFR)-fusion proteins to endomembranes. We showed that oleTMP mediated protein anchoring to endomembranes within minutes and could be reversed by the addition of free TMP. We also applied the endomembrane SLIPT system to artificially activate endomembrane Ras and inhibit the active nuclear transport of extracellular signal-regulated kinase (ERK), demonstrating its applicability for manipulating biological processes in living cells. We envision that the present oleTMP-based SLIPT system, which affords rapid and reversible control of protein anchoring to endomembranes, will offer a new unique tool for the study and control of spatiotemporally regulated cell signaling processes.

Effectiveness of retinoic acid (RA) in treating food allergy is not yet clear. Using an allergic mouse model, we examined the amelioration of the severity of food allergy by daily RA intake with allergen or without. Female Balb/c mice were systemically sensitized to egg white (EW) and alum by intraperitoneal injection. Sensitized mice were provided diets supplemented with 0% (non-treated group), 0.1% EW (allergen group), 0.0017% RA (RA group), or 0.1% EW plus 0.0017% RA (RA+allergen group) with 20% casein for 4 wk. Oral food challenge (OFC) and allergic biomarkers were quantified. The decrease in rectal temperature post-OFC was significantly suppressed in the RA and RA+allergen groups compared to those in the non-treated and allergen groups, respectivety. The plasma levels of ovalbumin-specific IgE, IgA and IgG1 at the study endpoint were higher in the allergen and RA+allergen groups than those in the non-treated and RA+allergen groups, respectivety. Plasma ovalbumin-specific IgG2a levels at the study endpoint were significantly higher in the RA+allergen group than those in the RA groups. The supernatant concentrations of interleukin-10 and interferon-γ in the cultured spleen lymphocytes were highest in the RA+allergen group compared to those in the other groups. Thus, continuous intake of RA under allergen exposure ameliorated the severity of food allergy in a mouse model with food allergy.

Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation., (© The Author(s) 2019. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

The ratio of syringyl (S) and guaiacyl (G) units in lignin has been regarded as a major factor in determining the maximum monomer yield from lignin depolymerization. This limit arises from the notion that G units are prone to C-C bond formation during lignin biosynthesis, resulting in less ether linkages that generate monomers. This study uses reductive catalytic fractionation (RCF) in flow-through reactors as an analytical tool to depolymerize lignin in poplar with naturally varying S/G ratios, and directly challenges the common conception that the S/G ratio predicts monomer yields. Rather, this work suggests that the plant controls C-O and C-C bond content by regulating monomer transport during lignin biosynthesis. Overall, our results indicate that additional factors beyond the monomeric composition of native lignin are important in developing a fundamental understanding of lignin biosynthesis.

We examined whether the stepwise oral immunotherapy (OIT) for 10 days ameliorates the severity of allergy and the biomarkers in an allergy mouse model. The OIT could not protect anaphylaxis symptoms after allergen challenges but promote the production of antibodies, especially allergen-specific IgA. It was suggested that this OIT influenced the function of immuno response against the allergen. Abbreviations: EW: egg white; IFC: intraperitoneal food challenge; IFN-γ: interferon-γ; IL: interleukin; OVA: ovalbumin; OM: ovomucoid; OFC: oral food challenge; OIT: oral immunotherapy.

Background: The objective of this study was to determine the required concentration of egg white (EW) in the diet to induce oral desensitization and/or immune tolerance within 4 weeks of oral immunotherapy (OIT) in an EW allergic mouse model., Methods: Female BALB/c mice were systemically sensitized to EW by intraperitoneal injections and subsequently subjected to oral allergen gavage. Sensitized mice were provided 4 weeks of OIT by supplementing with 0 (non-OIT), 0.01, 0.1, or 1% EW in a 20% casein diet. Nonsensitized mice served as the nonallergy group. We performed oral and intraperitoneal EW challenges, assessed vascular permeability in the dorsal skin, and measured allergic biomarkers., Results: The change in rectal temperature after oral challenge was not significantly different between the nonallergy and 1% EW groups, and the frequency of diarrhea in the 1% EW group was lower than that in the non-OIT group. The levels of plasma ovomucoid-specific IgE, IgA, and IgG2a in the 1% EW group at the study endpoint were significantly lower than those in the non-OIT group. IFN-γ and IL-10 secretions of spleen lymphocytes in the 1% EW group were significantly higher than those in the non-OIT group, and the percentage of CD4+Foxp3+ cells in the 1% EW group was higher than that in the non-OIT group., Conclusion: These results suggested that diet supplemented with 1% EW can induce oral desensitization and immune tolerance in the EW allergic mouse model., (© 2018 S. Karger AG, Basel.)

A novel renewable cathode made from earth abundant, low-cost materials can contribute to the intermittent storage needs of renewable energy-based society. In this work, we report for the first-time tannin from Nature as a cathode material. Our approach exploits the charge storage mechanism of the redox active quinone moiety. Tannins extracted from tree bark using environmental friendly aqueous solvents have the highest phenol content (5.56 mol g -1 ) among all the natural phenolic biopolymers, 5000 times higher than lignin. Tannins coupled with a conductive polymer polypyrrole acquire high specific capacitance values of 370 F g -1 at 0.5 A g -1 as well as excellent rate performance of 196 F g -1 at 25 A g -1 . Additionally, we employed carbonized wood as an electrode substrate to produce a sustainable electrochemical device with dramatically improved performance compared to conventional devices. The high surface area provided by the well-aligned, cellular porosity of wood-derived substrate combined with the high mobility of ions and electrons in the carbonized cell walls and deposited tannin can achieve an areal capacitance of 4.6 F cm -2 at 1 mA cm -2 , which is 1.5 times higher than activated wood carbon.

Commercial fungal cellulases used in biomass-to-biofuels processes can be grouped into three general classes: native, augmented, and engineered. Colorimetric assays for general glycoside hydrolase activities showed distinct differences in enzyme binding to lignin for each enzyme activity. Native cellulase preparations demonstrated low binding of endo- and exocellulases, high binding of xylanase, and moderate binding for β-D-glucosidases. Engineered cellulase formulations exhibited low binding of exocellulases, very strong binding of endocellulases and β-D-glucosidase, and mixed binding of xylanase activity. The augmented cellulase had low binding of exocellulase, high binding of endocellulase and xylanase, and moderate binding of β-D-glucosidase activities. Bound and unbound activities were correlated to general molecular weight ranges of proteins as measured by loss of proteins bands in bound fractions on SDS-PAGE gels. Lignin-bound high molecular weight bands correlated to binding of β-D-glucosidase activity. Whereas β-D-glucosidases demonstrated high binding in many cases, they have been shown to remain active. Bound low molecular weight bands correlated to xylanase activity binding. Contrary to other literature, exocellulase activity did not show strong lignin binding. The variation in enzyme activity binding between these three classes of cellulases preparations indicates that it is possible to alter the binding of specific glycoside hydrolase activities during the enzyme formulation process. It remains unclear whether or not loss of endocellulase activity to lignin binding is problematic for biomass conversion.

We have observed the 1-bond (199)Hg-(15)N J-coupling ((1)J((199)Hg,(15)N) = 1050 Hz) within the Hg(II)-mediated thymine-thymine base pair (T-Hg(II)-T). This strikingly large (1)J((199)Hg,(15)N) is the first one for canonical sp(2)-nitrogen atoms, which can be a sensitive structure-probe of N-mercurated compounds and a direct evidence for N-mercuration.

As part of our studies on the physiological and ecological function of caffeine, we investigated the effect of exogenously supplied caffeine on purine, pyrimidine and pyridine metabolism in rice seedlings. We examined the effect of 1 mM caffeine on the in situ metabolism of 14C-labelled adenine, guanine, inosine, uridine, uracil, nicotinamide and nicotinic acid. The segments of 4-day-old dark-grown seedlings were incubated with these labelled compounds for 6 h. For purines, the incorporation of radioactivity from [8-(14)C]adenine and [8-(14)C]guanine into nucleotides was enhanced by caffeine; in contrast, incorporation into CO2 were reduced. The radioactivity in ureides (allantoin and allantoic acid) from [8-(14)C]guanine and [8-(14)C]inosine was increased by caffeine. For pyrimidines, caffeine enhanced the incorporation of radioactivity from [2-(14)C]uridine into nucleotides, which was accompanied by a decrease in pyrimidine catabolism. Such difference was not found in the metabolism of [2-(14)C]uracil. Caffeine did not influence the pyridine metabolism of [carbonyl-14C]- nicotinamide and [2-(14)C]nicotinic acid. The possible control steps of caffeine on nucleotide metabolism in rice are discussed.

Accumulation and metabolism of purine alkaloids in leaves of maté (Ilex paraguariensis) were investigated. In winter, leaves accumulated caffeine but not theobromine, indicating that caffeine is the end product of purine alkaloid synthesis in maté. To elucidate the purine alkaloid metabolism in maté leaves, the metabolic fate of [8-(14)C]theobromine, [8-(14)C]theophylline, [8-(14)C]caffeine and [8-(14)C] xanthine was investigated in the leaf disks of young and mature leaves. In young maté leaves, significant amounts of theobromine and theophylline were utilized for caffeine biosynthesis, but the conversion was not observed in mature leaves. A small amount of theophylline was converted to theobromine. Practically no caffeine catabolism was detected in maté leaves during a 24 h-incubation. Catabolism of theobromine and theophylline via 3-methylxanthine was observed mainly in mature leaves. Xanthine was catabolised extensively via ureides in both young and mature leaves, but limited amounts are also utilized for the synthesis of theobromine, theophylline and caffeine. Possible pathways for the metabolism of purine alkaloids in maté leaves are discussed.

Some chalcones have been designed and synthesized using Claisen-Schmidt reactions as inhibitors of the ferredoxin and ferredoxin-NADP+ reductase interaction to pursue a new selective antimalaria agent. The synthesized compounds exhibited inhibition interactions between PfFd-PfFNR in the range of 10.94%-50%. The three strongest inhibition activities were shown by (E)-1-(4-aminophenyl)-3-(4-methoxyphenyl)prop-2-en-1-one (50%), (E)-1-(4-aminophenyl)-3-(2,4-dimethoxyphenyl)prop-2-en-1-one (38.16%), and (E)-1-(4-aminophenyl)-3-(2,3-dimethoxyphenyl)prop-2-en-1-one (31.58%). From the docking experiments we established that the amino group of the methoxyamino chlacone derivatives plays an important role in the inhibition activity by electrostatic interaction through salt bridges and that it forms more stable and better affinity complexes with FNR than with Fd.